扇贝、对虾基因组BAC文库构建及其重要功能基因的初步筛选和分析

基因组大片段BAC文库是进行生物遗传学和基因组学研究必不可少的基础工具。为了深入开展栉孔扇贝（Chlamys farreri）和凡纳滨对虾（Litopenaeus vannamei）基因组学研究、阐明其基因组的结构与功能、图位克隆重要功能基因、构建高密度物理图谱并最终实现与已有遗传连锁图的整合，本研究在植物基因组BAC文库构建技术的基础上，针对海洋生物的特点进行了大胆的改革与尝试，最终成功构建了C. ferrari和L. vannamei两种重要海水养殖动物的基因组BAC文库。 本文构建的栉孔扇贝基因组BAC文库，由BamHI文库和MboI文库构成。其中BamHI文库含有73,728个BAC克隆，空载率约为1%，平均插入片段约为110 kb，覆盖栉孔扇贝单倍体基因组约8倍；MboI文库共有7,680个克隆组成，平均插入片段大小约为145 kb，插入率为100%，覆盖栉孔扇贝单倍体基因组约1.1倍。两个栉孔扇贝基因组BAC文库共由81,408个克隆组成，平均插入片段约为113 kb，覆盖率约为栉孔扇贝单倍体基因组大小的9.1倍。 将栉孔扇贝基因组BAC文库的192个384微孔培养板中的73,728个BAC克隆以4 x 4点阵形式制备了高密度DNA薄膜，用于对感兴趣的基因及DNA序列的筛选。高密度DNA薄膜的覆盖率约为栉孔扇贝单倍体基因组的8.3倍。针对栉孔扇贝先天免疫系统通路的6个重要功能基因，根据栉孔扇贝cDNA序列以及异缘物种DNA序列设计了Overgo探针。利用Overgo探针对高密度DNA薄膜杂交筛选的结果显示，平均每个基因检测到7.3个潜在阳性克隆。 本研究所构建的凡纳滨对虾基因组HindIII酶切BAC文库共有102,528个BAC克隆，存放于267个384微孔培养板中，平均插入片段大小约为101 kb，空载率约为5%，覆盖L. vannamei单倍体基因组约5倍。将其中240个384微孔培养板中的92,160个BAC克隆以4 x 4的矩阵排列形式制作了5张高密度凡纳滨对虾DNA薄膜，约覆盖整个对虾单倍体基因组的4.5倍。针对6个与对虾免疫、生殖生理有关的重要功能基因设计了Overgo探针，杂交筛选出20个阳性克隆，平均每个基因有3.3个潜在阳性克隆。 以上筛选结果不仅为进一步研究这些功能基因的结构与功能、表达与调控，揭示它们在扇贝和对虾以及其他近缘种的免疫系统、抗逆和生殖生理过程中的作用机理打下了基础，同时也间接验证了栉孔扇贝和凡纳滨对虾基因组BAC文库将成为基因筛选、基因的结构与功能分析、基因图位克隆、物理图谱构建以及大规模全基因组测序等方面的有力工具。

温度盐度对栉孔扇贝Chlamys farreri胚胎和幼虫的影响

本文在实验室条件下，系统地研究了温度盐度对栉孔扇贝胚胎和幼虫的单一和组合效应。受精卵孵化的适宜温度范围为16.0-22.0 ℃，适宜盐度范围为27.5-32.5‰。随着胚胎发育，胚体对度盐度的适应范围增广。胚胎孵化的适宜温－－盐区域为16.0 ℃／27.2‰-24.4 ℃/33.6‰。水温超过26.0 ℃时，幼虫存活率显著下降，其生长的适宜温度范围为16.0-26.0℃。幼虫存活的适宜盐度范围为27.0-42.0‰，生长的适宜盐度范围为27.0-39.0‰。在120小时实验中，幼虫存活的适宜温－－盐区域是10.0 ℃/16.3‰-27.0 ℃/42.0‰，生长的适宜的温－－盐区域是21.5 ℃/26.1‰-27.0 ℃/36.0‰。在240小时实验中，幼虫存活的适宜温－－盐区域是10.0 ℃/23.4‰-26.9 ℃/42.0‰，生长的适宜的温－－盐区域是14.2 ℃/20.5‰-27.6 ℃/38.8‰。温度盐度对胚胎和幼虫的影响都很大，相比较而言，盐度的影响大于温度。温－－盐对胚胎发育和幼虫存活的组合效应极其显著。随着幼虫的生长发育，温－－盐对幼虫生长的组合效应逐渐表现出显著性，幼虫生长的适应范围增大。

Fluorescence Characterization of Dissolved Organic Matter in East China Sea

Sea water samples were collected in the East China Sea in March and April, 2005, and three-dimensional fluorescence of dissolved organic matter was measured by fluorescence excitation-emission matrix spectroscopy. The position, number and intensity of fluorescence peak in the spectra and the relations of the peaks were analyzed to determine the type, distribution and origin of the fluorescence dissolved organic matter. Seven types of fluorescence peaks were detected from the samples. There are protein-like fluorescence peaks B with Ex(max)/Em(max) = 275/300 nm, D with Ex(max)/Em(max) = 225/295-305 nm, T with Ex(max)/Em(max) = 280/345 nm, and S with Ex(max)/Em(max) = 225-240/320-350 nm, two humic-like peaks A with Ex(max)/Em(max) = 250-255/410-455 nm and C 335-345/410-440 ran, and marine humic peak M with Ex(max)/Em(max) = 305 nm/400-420 nm. Peaks B, S and A appeared in all surveyed area. Peaks T and D appeared in the north of the surveyed area. Peaks M and C only appeared in a few stations. In the surface layer, the source of the fluorescence dissolved organic matter might be the fresh water outflow of the Yangtze River, while the fluorescence dissolved organic matter in the middle layer had double sources from the Yangtze River and the phytoplankton. The good correlationships of different fluorescence peaks showed the same source or some relationship between the protein-like and the humic-like fluorescence dissolved organic matter.

Fluorescence Characterization of Dissolved Organic Matter in the East China Sea after Diatom Red Tide Dispersion

Fluorescence excitation-emission spectroscopy (EEMS) was employed to analyze the 3-dimensional fluorescence of dissolved organic matter in the East China Sea after diatom red tide dispersion. The relationships between fluorescence peak intensity, and salinity and chlorophyll-a were discussed. The centers of protein-like fluorescence peaks dispersed at Ex(max)/Em(max) = 270-280/290-315 nm (Peak B), 220-230/290-305 nm (Peak D), 230-240/335-350 nm(Peak S)and 280/320 nm(Peak T). Two humic-like peaks appeared at 255-270/435-480 nm (Peak A) and 330-350/420-480 nm(Peak C). High tyrosine-like intensity was observed in diatom red tide dispersion area, and tryptophan-like fluorescence was also found which was lower. High FIB/FIS showed that diatom red tide produced much tyrosine-like matter during dispersion. Peaks S, A and C had positive correlation with one another, and their distributions were similar, which decreased with distance increasing away from the shore. Good negative correlations between peaks S, A and C and salinity suggested that Jiangsu-Zhejiang coastal water was the same source of then-L Correlations between fluorescence peak intensity and chlorophyll-a were not remarkable enough to clear the relationship between fluorescence and living algal matter. It was supposed that the living algal matter contributed little to the fluorescence intensity of algal dispersion seawater.

Studies on nitric oxide synthase activity in haemocytes of shrimps Fenneropenaeus chinensis and Marsupenaeus japonicus after white spot syndrome virus infection

The nitric oxide synthase (NOS) activity in the haemocytes of shrimps Fenneropenaeus chinensis (Osbeck) and Marsupenaeus japonicus (Bate) was Studied after white spot syndrome virus (WSSV) infection to determine its characteristics in response to virus infection. First, the NOS activity in haemocytes of shrimps was determined by the means of NBT reduction and changes in cell conformation. And the variations of NOS activity in shrimps after challenge with WSSV intramuscularly were evaluated through the analysis Of L-citrulline and total nitrite/nitrate (both as NO derivates) concentrations. The result showed that NOS activity in the haemocytes of F chinensis increased slightly from 0 to 12 h postchallenge, indicated by the variations Of L-Citrulline (from 11.15 +/- 0.10 to 12.08 +/- 0.64 mu M) and total nitrite/nitrate concentrations (from 10.45 +/- 0.65 to 12.67 +/- 0.52 mu M). Then it decreased sharply till the end of the experiment (84 h postchallenge), the concentrations Of L-Citrulline and total nitrite/nitrate at 84 It were 1.58 +/- 0.24 and 2.69 +/- 0.70 mu M, respectively. The LPS-stimulated NOS activity kept constant during the experiment. However, in M. japonicus, the NOS activity kept increasing during the first 72 It postchallenge, the concentrations Of L-Citrulline and total nitrite/nitrate increased from 7.82 +/- 0.77 at 0 h to 10.79 +/- 0.50 mu M at 72 h, and from 8.98 +/- 0.43 at 0 h to 11.20 +/- 0.37 mu M at 72 h, respectively. Then it decreased till the end of the experiment (216 h postchallenge), and the concentrations of L-Citrulline and total nitrite/nitrate at 216 h were 5.66 +/- 0.27 and 4.68 +/- 0.16 mu M, respectively. More importantly, an apparent increase of I-PS-stimulated NOS activity was observed in M japonicus at 48 h postchallenge, which was about 4 times higher than that in the control group of health shrimps. In correspondence with the difference of NOS activity between the two species of shrimps, the Cumulative mortalities of the shrimps were also different. All shrimps of F. chinensis in the mortality experiment died in 66 h, much more quickly than M. japonicus, Whose accumulative mortality reached 100% after 240 h. Data here reported let us hypothesize that NOS activity in the haemocytes of shrimps F chinensis and M. japonicus responses to WSSV infection differently, and this might be one of the reasons for the different susceptibility of F chinensis and M. japonicus to WSSV infection. (c) 2005 Elsevier Inc. All rights reserved.

Photocatalytic degradation of CI Acid Blue 80 in aqueous suspensions of titanium dioxide under sunlight

The dye C.I. Acid Blue 80 (AB80) was easily degraded by TiO2-P25 assisted photocatalysis in aqueous dispersion under irradiation of sunlight. The optimal reaction conditions were [TiO2] = 2.0 g/L, pH = 10, [H2O2] = 5 mmol/L. The photocatalytic reaction followed pseudo-first order kinetics. The adsorption of AB80 onto TiO2 was in accord with Langmuir equation.

In vivo antitumor and immunomodulation activities of different molecular weight Lambda-Carrageenans from Chondrus ocellatus

lambda-Carrageenan is a sulfated galactan isolated from some red algae and have been reported to have many kinds of biological activities. lambda-Carrageenan from Chondrus ocellatus, an important economic alga in China and many other parts of the world, was degraded by microwave, and obtained five products that have different molecular weight: 650, 240, 140, 15, 9.3 kDa. Analytical results confirmed that microwave degradation might not change the chemical components and structure of polysaccharides under certain condition. In this study, tumor-inhibiting activities, weight of immune organ, nature killer cells activity, lymphocyte proliferation ratio and pathological slice of spleen and tumor cells from the control group and lambda-carrageenan-treated mice of transplanted S 180 and H22 tumor were investigated. The results indicated that the five lambda-carrageenan samples all showed antitumor and immunomodulation activities in different degree. Molecular weight of polysaccharides had notable effect on the activities. In addition, their antitumor and immunomodulation have some relevance and the five lambda-carrageenans probably inhibited tumor by means of activating the immunocompetence of the body. Among all the experiment results, samples with the highest activities are PC4 and PC5 whose molecular weight are 15 and 9.3 kDa. (C) 2003 Elsevier Ltd. All rights reserved.

藏药达乌里秦艽中环烯醚萜苷的HPLC测定

采用HPLC法同时测定了藏药达乌里秦艽中龙胆苦苷(gentiopicroside)、獐牙菜苦苷(swertiamarin)、獐牙菜苷(sweroside)和落干酸(loganic acid)4种主要环烯醚萜苷类成分,利用在线质谱(MS)进行了定性分析,并与同属藏药麻花艽进行了比较。色谱柱为Eclipse XDB-C_8(4.6 mm×150 mmi.d.,5μm),流动相A:体积分数5%乙腈(含10 mmol甲酸)水溶液,流动相B:体积分数95%乙腈水溶液,以流动相A在0～15 min内由100%线性减至90%,15～20 min内保持90%不变线性梯度洗脱。流速1.0 mL/min,检测波长240 nm,柱温30℃。结果表明,4种成分均达到了基线分离,具有较好的线性关系和回收率。本法可作为达乌里秦艽药材有效成分的测定方法,为其有效成分的进一步开发利用提供依据。

液相色谱/质谱大气压化学电离源鉴定深海鱼油中长链不饱和脂肪酸

以1,2-苯并-3,4-二氢咔唑-9-乙基苯磺酸酯为衍生化试剂,在充氮的气氛下对鱼油进行皂化处理,所得皂化产物经正己烷萃取处理后进行柱前衍生化,再以HPLC/MS分离和鉴定。通过对长链脂肪酸分子的标记处理,其衍生物分子在质谱分析中呈现出双键位置的规范信息。通过建立模型计算式,借助不饱和脂肪酸的分子离子峰和特征碎片离子峰的质量数,计算不饱和的碳碳双键位置。共鉴定出23种脂肪酸。结果表明深海鱼油主要由C12-C22的脂肪酸组成,多不饱和脂肪酸含量占67.08%（峰面积百分比,下同）,其中C16∶19-十六碳烯酸（11.7%）;C16∶44,7,10,13-十六碳四烯酸（2.91%）;C18∶112-十八碳烯酸（11.1%）;C18∶46,9,12,15-十八碳四烯酸（3.62%）;C20∶113-二十碳烯酸（1.21%）;C20∶55,8,11,14,17-二十碳五烯酸（16.71%）;C22∶62,5,8,11,14,17-二十二碳六烯酸（10.53%）。所建立的方法为不饱和脂肪酸碳链中双键位置的确定提供了新的技术手段。

藏药麻花艽中四种苦苷类化学成分的HPLC测定

建立了藏药麻花艽中龙胆苦苷（gentiopicroside）、落干酸（loganic acid）、獐牙菜苦苷（swertiamarin）和獐牙菜苷（sweroside） 4种苦苷类成分的HPLC分析方法. 色谱柱为Eclipse XDB-C8 （4.6 mm i.d. ×150 mm, 5 μm）, 流动相为体积分数5%乙腈＋10 mmoL甲酸～95%乙腈水溶液, 检测波长为240 nm. 结果表明, 该方法具有良好的线性关系和回收率, 为麻花艽中苦苷类成分的进一步开发利用提供了科学依据.

HPLC法测定野生与栽培藏药麻花艽中4种环烯醚萜苷类成分的含量

目的：对野生和栽培藏药麻花艽中龙胆苦苷、落干酸、獐牙菜苦苷和獐牙菜苷4种苦苷类成分进行高效液相色谱的含量测定，并比较分析它们之间的差异。方法：采用Eelipse XDB-C_8色潜柱(4.6 mm*150 mm，5 μm)，流动相A为95％乙腈水溶液，B为5％乙腈(含10 mmol•L~(-1)的甲酸)水溶液，A在0-20min内比例由0-100％进行线性洗脱，流速1.0 mL•min~(-1)，检测波长240 nm，柱温30℃。结果：4种成分均达到基线分离，龙胆菁苷、落干酸、獐牙菜苦苷、獐牙菜苷的线性范围分别为0.60～19•20μg(r=0.9999)，0.24～7.68μg(r=O.9999)，0.38-12.02μg(r=O.9999)，0.05～1.66μg(r=0.9999)；回收率分别为99.73％，98.13％，98.45％，96.22％。结论：栽培藏药麻花艽中苦苷类成分的含量已经接近或超过野生种的水平，可初步代替野生药材人药。

青藏铁路沿线高寒草甸植物群落特征对人为干扰梯度的响应——以风火山高山嵩草草甸为例

选择风火山地区受到人为干扰的高寒草甸，对处于不同干扰梯度的高寒草甸植物群落，用样带法进行调查．共调查5条样带，50个样方．统计植物的科属组成，计测其群落盖度、频度指标以及物种丰富度指数、多样性指数及均匀度指数等．对不同干扰梯度上的植物群落结构及其物种组成特点进行分析，结果表明：青藏铁路沿线的高寒草甸随着人为干扰的强度的增加，植物群落的科、属、种组成趋向单一；群落中起显著作用的物种趋向是高山嵩草→高山嵩草和矮嵩草→早熟禾和扇穗茅→早熟禾→羊茅和早熟禾；按照植物种群的重要值对干扰梯度的响应，该地区植物种群可被划分为3类响应模式，即忍耐型种、敏感型种和扩展型种．随着干扰强度的增加，物种丰富度指数和物种多样性随着干扰增加而降低．干扰梯度与有机质含量、全氮、水解氮、全磷、速效磷均呈显著相关，但是与全钾和速效钾相关关系不明显．

不同土壤水分含量下高寒草地CH4释放的比较研究

2003年6月30日～9月4日,利用密闭箱-气相色谱法,对发育于不同水分状况下的灌丛草甸(GC)、矮嵩草草甸(AC)、藏嵩草草甸(ZC)和季节性湿地(SD)的CH4释放速率进行了比较研究.结果表明,观测期间,季节性湿地处于淹水状态,其它三种土壤平均水分含量分别为39.6%(GC)、38.4%(AC)、65.9%(ZC),而CH4平均释放速率分别为-0.031±0.030(GC)、-0.026±0.018(AC)、1.103±0.240(ZC)和6.922±4.598 mg•m-2•h-1(SD),随着土壤水分含量的增加,高寒草地土壤CH4释放由吸收转为排放,表现出与土壤湿度很好的一致性.矮嵩草草甸不同处理CH4吸收强度AC＜AJ＜AL,它们之间的差异除与土壤水分有关,还可能与处理引起的CH4传输途径不同有关.实验期间,矮嵩草草甸和灌丛草甸土壤-植物系统分别吸收CH438.69和46.13 mg•m-2,是大气温室气体CH4的弱汇,藏嵩草草甸和季节性湿地则是大气温室气体CH4的源,分别排放CH4 1.641和10.30 g•m-2.

高寒草甸群落植物多样性和初级生产力沿海拔梯度变化的研究

对不同海拔梯度高寒草甸群落植物多样性和初级生产力关系的研究结果表明:1)不同海拔梯度上,中间海拔梯度群落植物多样性最高,即物种丰富度、均匀度和多样性最大;2)不同海拔梯度上,群落生产力水平和物种丰富度中等时,物种多样性最高;3)随着海拔的逐渐升高,地上生物量逐渐减少;4)地下生物量具有"V"字形季节变化规律,在牧草返青期和枯黄期地下生物量最大,7月最小,且地下生物量主要分布在0～10 cm的土层中.地下生物量垂直分布呈明显的倒金字塔特征.

Distribution and species diversity of plant communities along transect on the Northeastern Tibetan plateau

The distribution and species diversity of plant communities along a 600 km transect through the northeastern Tibetan Plateau (32 degrees 42'-35 degrees 07' N, 101 degrees 02'-97 degrees 38' E) with altitudes from 3255 to 4460 m are described. The transect started from the Youyi Bridge of Banma through Dari, Maqin and Maduo to Zaling Lake. The data from 47 plots along the transect are summarized and analyzed. The mean annual temperature, the mean annual rainfall and the length of growing season decreases from 2.6 to -4.5 degrees C, from 767.2 to 240.1 mm, from 210 to 140 days, respectively, along the transect from the southeastern Banma to northwestern Zaling Lake. The number of vascular plant species recorded in 47 plots is 242 including 2 tree, 34 shrub, 206 herb species. Main vegetation types on the transect from southeast to northwest are: Sabina convallium forest, Picea likiangensis forest, Pyracantha fortuneana + Spiraea alpina shrub, Hippophae neurocarpu shrub, Sibiraea angustata + Polygonum viviparum shrub, Stellera chamaejasme herb meadow, Potentilla fruticosa + Salix obscura + Carex sp. Shrub, Kobresia capillifolia meadow, P. froticosa + Kobresia humilis shrub, Caragana jubata + S. obscura shrub, Kobresia tibetica meadow, Kobresia pygmaea meadow, K. pygmaea + Stipa purpurea steppe meadow, Stipa purpurea steppe. Plant richness and diversity index all showed a decreasing trend with increasing of elevation along transect from southeast to northwest. Detailed information on altitudinal ranges and distribution of the alpine vegetation, vascular flora and environments over the alpine zone at northeastern Tibetan Plateau provides baseline records relevant to future assessment of probable effects of global climate changes.